Dengue virus (DENV) is a mosquito-transmitted virus from the genus Flavivirus. It is the most common cause of mosquito-borne viral diseases in tropical and subtropical regions around the world, and is expanding in geographic range and also in disease severity. The virus is a small, enveloped, icosahedral virus, with positive strand RNA of 11,000 nucleotides (Thaisomboonsuk, et al., Characterization of dengue-2 virus binding to surfaces of mammalian and insect cells. Am J Trop Med Hyg. 2005 April; 72(4):375-83). There are four distinct serotypes of dengue that cause similar disease symptoms, serotypes 1-4 (DENV-1, DENV-2, DENV-3, and DENV-4) that cocirculate in many areas of the world and give rise to sequential epidemic outbreaks when the number of susceptible individuals in the local population reaches a critical threshold and weather conditions favor reproduction of the mosquito vectors Aedes aegypti and Aedes albopictus. 
2.5 billion people living in regions where dengue is endemic are at risk of infection (Mackenzie, et al., Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat. Med. 2004 December; 10((12 Suppl):S98-S109; WHO 2012. Dengue and severe dengue. Fact sheet no. 117 last updated Sep. 13, 2018). Exposure to Dengue virus typically results in symptoms 3 days to 2 weeks after exposure. Approximately 50 to 100 million people per year are infected with DENV. DENV infections may be asymptomatic, but most often manifest as dengue fever (DF), a self-limited disease. An estimated 500,000 people, many of them children, are hospitalized annually with severe dengue symptoms, including dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (WHO 2012. Dengue and severe dengue. Fact sheet no. 117 last updated Sep. 13, 2018; Gubler, Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. Trends Microbiol. 2002 February; 10(2):100-103). Dengue infection symptoms include severe headache, occular pain; muscle, joint, and hone pain; macular or maculopapular rash; and varying levels of hemorrhagic response.
Infection with one serotype confers lifelong homotypic immunity, that is protective against that same serotype. The infection causes a cross-reactive antibody response against the other serotypes (and other flaviviruses as well). However, the immunity only provides short term (approximately three to six months) cross protection against heterotypic serotypes (Sabin, Research on dengue during World War II. Am. J. Trop. Med. Hyg. 1952 January; 1(1):30-50). Secondary, or more, DENV infections tend to produce broadly neutralizing response. Low levels of neutralizing antibodies, cross-reactive but nonneutralizing antibodies, or both from previous infections bind virions of other serotypes and target them to Fc receptors on macrophages and certain other cell types, enhancing infection of these cells (Halstead, et al., Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody. J. Exp. Med. 1977 Jul. 1; 146(1):201-17). DENV imposes one of the largest social and economic burdens of any mosquito-borne viral pathogen. There is no specific treatment for infection, and control of dengue virus by vaccination has proved elusive. However, the risk of severe disease is greatest during secondary, heterotypic infections in subjects with more than one circulating serotype. An increasing problem for public health officials has been the occurrence of severe complications arising from dengue viral infection. Both dengue hemorrhagic fever (DHF) and shock syndromes (DSS) are clinical outcomes related to the presence of pre-existing immunity to a heterologous dengue virus serotype. The presence of these cross-reactive and nonneutralizing antibodies also correlated with severe disease outcome (DHF/DSS) in several studies (Halstead, Pathogenesis of dengue: challenges to molecular biology. Science. 1988 Jan. 29; 239(4839):476-81; Kliks, et al., Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. Am. J. Trop. Med. Hyg. 1988 March; 38(2):411-9; Vaughn, et al., Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J. Infect. Dis. 2000 January; 181(1):2-9). Higher levels of viremia are associated with the development of DHF (Vaughn, et al., Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J. Infect. Dis. 2000 January; 181(1):2-9; Vaughn, et al., Dengue in the early febrile phase: viremia and antibody responses. J. Infect. Dis. 1997 August; 176(2):322-30). A preponderance of antibodies that recognize neutralizing epitopes will lead to virus clearance and reduced symptoms, while an abundance of antibodies that recognize enhancing epitopes will lead to more severe disease. Antibody-dependent enhancement (ADE) is an increase in viral infection as a result of antibody-mediated cellular entry, is common among flaviviruses, and has been shown to decrease as viral particles remain extracellularly with antibodies, i.e. virus particles exposed to antibodies experience time-dependent loss of infectivity even when exposed to partially neutralizing antibodies, which is not attributed to increased antibody binding (Dowd, et al., A dynamic landscape for antibody binding modulates antibody-mediated neutralization of West Nile virus. PLoS Pathog. 2011 June; 7(6):e1002111). This antibody-dependent enhancement effect may also explain the sequential nature of epidemic outbreaks, as well as the severe disease seen in infants as maternal antibodies wane (Halstead, Pathogenesis of dengue: challenges to molecular biology. Science. 1988 Jan. 29; 239(4839):476-81; Kliks, et al., Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. Am. J. Trop. Med. Hyg. 1988 March; 38(2):411-9; Simmons, et al., Maternal antibody and viral factors in the pathogenesis of dengue virus in infants. J. Infect. Dis. 2007 Aug. 1; 196(3):416-24).
Dengue Haemorrhagic Fever is initially characterized by a minor febrile illness lasting 3-5 days. The patient may deteriorate at defervescence into the next phase of the syndrome with hemostatic disorders, and increased vascular permeability frequently accompanied by internal bleeding and shock. As many as 1.5 million children are reported to have been hospitalized with 33,000 deaths from this syndrome since it was first recognized in the Philippines and Thailand in the 1950s. DHF/DSS has since continued to persist, and outbreaks can pose major problems to public health in many countries. Unfortunately, the pathogenesis of DHF/DSS is not completely understood. Epidemiological studies have shown that the presence of cross-reactive antibodies correlates with a more severe disease outcome during subsequent infections with a different serotype. The mechanism for this effect appears to be an antibody-dependent enhancement of infection of macrophage and macrophage-like cells that express Fc receptors. These cells are normally not infected efficiently by dengue, but become highly infectable in the presence of dengue virus binding antibodies that then target the virus particles directly to the macrophages through the interaction of the antibody heavy chains and the cellular Fc receptors.
Like other members of the genus Flavivirus, DENV has a lipid envelope and a positive-strand RNA genome that codes for a single large polyprotein that encodes 3 structural proteins- the capsid (C) protein, the membrane (M) protein, and the envelop (E) protein- and 7 nonstructural proteins, including proteases and RNA polymerase. This polyprotein is cleaved into separate segments to form the capsid (C), premembrane (prM/M), and envelope (E) structural proteins and enzymatic components required for viral replication and transmission (Dengue and dengue hemorrhagic fever: history and current status. Gubler, Goode. Eds. (Novartis Foundation (2006)).
The E glycoprotein assembles as a dimer on the viral surface, and possesses domains, DI, DII, and DIII, as well as a transmembrane domain (Modis, et al., A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc. Natl. Acad. Sci. U.S.A 2003 Jun. 10; 100(12):6986-91; Rey, et al., The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature. 1995 May 25; 375(6529):291-8). At one end of the molecule is the fusion loop within DII, and at the other end is DIII, which is involved in host cell binding (Crill, et al., Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J. Virol. 2001 August; 75(16):7769-73). E protein is believed to facilitate cell binding to surface receipts, like heparin sulfate, resulting in endocytosis or fusion with the plasma membrane itself. Studies suggest the virus is uptaken by antibody-dependent Fc receptor endocytosis or trypsin-sensitive receptor-based endocytosis, whereby the virus is released into the cell via a fusion loop, found in domain DII of the E protein (Thaisomboonsuk, et al., Characterization of dengue-2 virus binding to surfaces of mammalian and insect cells. Am J Trop Med Hyg. 2005 April; 72(4):375-83). During the infection process, the fusion loop is projected outward by a structural rearrangement of the E protein, resulting in the fusion loop “harpooning” into the target cell membrane. This interaction is critical for the subsequent membrane fusion step, mediated by a further E protein movement that pulls the cell and virus membranes together. Low pH causes a conformation change in the E protein, exposing domains in or around the fusion loop, which interact with host receptors, including CD209, Rab 5, GRP 78, and the mannose receptor, to mediate entry into a cell. During viral packaging, the external E glycoprotein is physically arranged in a herringbone pattern as a series of 90 homodimers on the outer surface of the mature virus particle (Kuhn, et al., Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell. 2002 Mar. 8; 108(5):717-25). On immature particles, the prM protein lies over the E protein and serves to protect the virus particle from undergoing premature fusion or inactivation within the secretory pathway of the host cell. prM is subsequently cleaved by a host protease to release the ectodomain and allow viral maturation (Yu, et al., Structure of the immature dengue virus at low pH primes proteolytic maturation. Science. 2008 Mar. 28; 319(5871):1834-7). Upon infection and entry of DENV into the acidic environment of the endosome, the E proteins undergo a conformational change and reassemble into 60 trimers with their fusion loops forming the tip of a trimeric spike oriented to insert into the endosomal membrane within the target cell. Subsequent reconfiguration of the E protein trimers results in fusion of the viral membrane and target cell endosomal membrane to facilitate release of the viral contents into the cytoplasm (Harrison, Viral membrane fusion. Nat. Struct. Mol. Biol. 2008 July; 15(7):690-8; Heinz, et al., Flavivirus structure and membrane fusion. Adv. Virus Res. 2003; 59:63-97; Zhang, et al., Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nat. Struct. Biol. 2003 November; 10(11):907-12).
Monoclonal antibodies (MAbs) have been used to further elucidate important epitopes. However, to date, most anti-DENV monoclonal antibodies are of murine origin (mMAbs), generated from mice (Crill, et al., Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J. Virol. 2001 August; 75(16):7769-73; Halstead, Neutralization and antibody-dependent enhancement of dengue viruses. Adv. Virus Res. 2003; 60:421-67; Sukupolvi-Petty, et al., Structure and function analysis of therapeutic monoclonal antibodies against dengue virus type 2. J. Virol. 2010 September; 84(18):9227-39). mMAbs may not accurately represent the human antibody response to DENV, as mice do not experience human disease other than a transitory viremia and produce an antibody response with more limited diversity and typically lower-affinity antibodies than humans. Recent studies with human monoclonal anti-DENV antibodies (hMAbs) have highlighted both similarities and major differences between the behavior of sera from convalescent DENV patients and purified hMAbs.
Further, studies have attempted to determine the human antibody response against dengue virus by characterizing human anti-dengue monoclonal antibodies. The nature of the human antibody response to DENV is likely to play a dominant role in defining the outcome of infection. Studies with sera from convalescent DENV patients have yielded conflicting information regarding the human antibody response and the epitopes that these antibodies target.
In the work of Schieffelin et al., three antibodies that targeted the E protein were isolated from a single donor (Schieffelin, et al., Neutralizing and non-neutralizing monoclonal antibodies against dengue virus E protein derived from a naturally infected patient. Virol. J. 2010 Feb. 4; 7:28). All three antibodies were cross-reactive with at least two DENV serotypes, one was neutralizing, and all were able to enhance DENV infection. Dejnirattisai et al. reported that in a panel of hMAbs from seven donors, the majority of the antibody response was against prM and was very poorly neutralizing but highly enhancing (Dejnirattisai, et al., Cross-reacting antibodies enhance dengue virus infection in humans. Science. 2010 May 7; 328(5979):745-8). Beltramello et al. described a wide variety of hMAbs from five DENV patients (Beltramello, et al., The human immune response to Dengue virus is dominated by highly cross-reactive antibodies endowed with neutralizing and enhancing activity. Cell Host Microbe. 2010 Sep. 16; 8(3):271-83). They included hMAbs against prM, as well as E. However, in contrast to the findings of Dejnirattisai, et al., half of the prM hMAbs reported by Beltramello et al. showed substantial neutralization activity (Beltramello, et al., The human immune response to Dengue virus is dominated by highly cross-reactive antibodies endowed with neutralizing and enhancing activity. Cell Host Microbe. 2010 Sep. 16; 8(3):271-83). Among the hMAbs recognizing E, Beltramello et al. described antibodies targeting DI/II and DIII. The DIII hMAbs were very highly neutralizing and included serotype-specific and cross-reactive examples. The neutralization activities of the DI/II hMAbs were more diverse and included nonneutralizing, serotype-specific neutralizing, and cross-neutralizing examples. Two of the cross-neutralizing DI/II hMAbs were mapped to the fusion loop using West Nile virus (WNV) E protein mutants. However, antibodies to this flavivirus epitope are typically conformation-sensitive (Lai, et al., Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II. J. Virol. 2008 July; 82(13):6631-43)
de Alwis et al. reported that after primary infection most hMAbs were cross-reactive and weakly neutralizing and that many bound to prM (de Alwis, et al., In-depth analysis of the antibody response of individuals exposed to primary dengue virus infection. PLoS Negl. Trop. Dis. 2011 June; 5(6):e1188). Using a modified screening procedure, they were able to detect rare DIII hMAbs that were serotype specific and strongly neutralizing. Recently, de Alwis et al. reported that the majority of antibodies in human sera bound to intact virions, not monomeric E (de Alwis, et al., Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions. Proc. Natl. Acad. Sci. U.S.A. 2012 May 8; 109(19):7439-44). They found that though abundant in human sera, cross-reactive antibodies did not contribute to neutralization and that type-specific antibodies were responsible for potent neutralization. These findings were confirmed with 3 hMAbs that were isolated by first screening for antibodies that bound to intact virions and then screening for a subset of antibodies that were potently neutralizing. They generated escape mutants and mapped the mutations to the quaternary epitopes containing contacts on two different E proteins in the hinge region between DI and DII.
Interestingly, while one of the predominant epitopes recognized by human serum antibodies appears to include the fusion loop and adjacent regions (Lai, et al., Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II. J. Virol. 2008 July; 82(13):6631-43; Lin, et al., Analysis of epitopes on dengue virus envelope protein recognized by monoclonal antibodies and polyclonal human sera by a high throughput assay. PLoS Negl. Trop. Dis. 2012 January; 6(1):e1447), one study reported that these fusion loop antibodies are nonneutralizing (Lai, et al., Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II. J. Virol. 2008 July; 82(13):6631-43). He et al. tested the ability of patient sera to block binding of DENV serotype 2 (DENV-2) to Vero cells and reported that neutralization occurred primarily by blocking cell attachment, suggestive of a major role for antibodies targeting DIII (He, et al., Antibodies that block virus attachment to Vero cells are a major component of the human neutralizing antibody response against dengue virus type 2. J. Med. Virol. 1995 April; 45(4):451-61). In contrast, Wahala et al. subsequently reported that human antibodies directed toward epitopes other than DIII (presumably DI/III) are primarily responsible for neutralization (Wahala, et al., Dengue virus neutralization by human immune sera: role of envelope protein domain III-reactive antibody. Virology. 2009 Sep. 15; 392(1):103-13).
Thus, multiple questions remain about the nature of the antibody balance, including which epitopes are most important for neutralization versus enhancement and whether these are distinct or overlapping epitopes. One of the conclusions to come out of the human studies is that the dominant human antibody response against the dengue virus surface proteins, membrane (prM and M) and envelope (E, soluble envelope protein, sE), is non-neutralizing and cross reactive against the four serotypes of dengue. These non-neutralizing, cross-reactive antibodies are the primary cause of the antibody dependent enhancement of disease. These studies with hMAbs emphasize the complexity of the human antibody response against DENV and highlight the importance of further examination of the roles of different epitopes in prM, in E protein DI/II (either the fusion loop or the hinge region), and in DIII and the mechanisms by which different antibodies neutralize DENV infection. For instance, an affected stage of viral entry—virus binding to the cell surface versus fusion between the viral envelope and endosomal membrane—has never been identified for any neutralizing hMAb.
Ongoing research is focused on potential vaccines to dengue viral infection. In 2015, Sanofi Pasteur was approved to release Dengvaxia, a dengue vaccine based on an attenuated yellow fever strain possessing the premembrane (PrM) and envelope (E) genes of four dengue virus serotypes, i.e. an attenuated tetravalent dengue vaccine. Prior work has shown yellow fever vaccine 17D (YF-17D) induces an efficient immune response against infection, which usually begins by around 10 days after vaccination in 95% of vaccine recipients and can last up to 35 years post-vaccination (Poland, et al., Persistence of neutralizing antibody 30-35 years after immunication with 17D yellow fever vaccine. Bull World Health Organ. 1981; 59(6):895-900; Monath, et al., Pathogenesis and pathophysiology of yellow fever. Adv Virus Res. 2003; 60:343-95; Niedrig, et al., Assessment of IgG antibodies against yellow fever virus after vaccination with 17D by different assays: neutralization test, haemagglutination inhibition test, immunofluorescence assay and ELISA. Trop Med Int Health. 1999; 4(12):867-71). Studies suggests YF-17D infect dendritic cells and activate multiple Toll-like receptors, resulting in a strong immunogenic response (Muyanja, et al., Immune activation alters cellular and humoral responses to yellow fever 17D vaccine. J Clin Invest. 2014 Jul. 1; 124(7):3147-58; Querec, et al., Systems biology approach predicts immunogenticity of the yellow fever vaccine in humans. Nat Immunol. 2008; 10(1):116-25; Barba-Spaeth, Live attenuated yellow fever 17D infects human DCs and allows for presentation of endogenous and recombinant T cell epitopes. J Exp Med. 2005; 202(9):1179-84; Martins, et al., Activation/modulation of adaptive immunity emerges simultaneously after 17DD yellow fever first-time vaccination: is this the key to prevent severe adverse reactions following immunization? Clin Exp Immunol. 2007; 148(1):90-100; Mandl, et al., Distinctive TLF? Signaling, type I IFN production, and attenuated innate and adaptive immune responses to yellow fever virus in a primate reservoir host. J Immunol. 2011; 186(11):6406-16). Concerns regarding the possibility of vaccine components eliciting enhancing antibody responses, as opposed to protective responses, have been a major concern in designing and testing vaccines to protect against dengue infections. There is thus a need for a vaccine that may be effective against different serotypes and which does not enhance the course of the DENV infection. In fact, Dengvaxia was found to provide only partial protection, and was found to worsen disease symptoms in some individuals whom had no prior dengue viral exposure, resulting in some countries withdrawing the vaccine.
Numerous factors can affect vaccine responses, including genetic background, gender, age, and environmental conditions (Monath, et al., Comparative safety and immunogenicity of two yellow fever 17D vaccines (ARILVAX and YF-VAX) in a phase III multicenter, double-blind clinical trial. Am J Trop Me Hyg. 2002; 66(5):533-41; Monath, et al. Yellow fever 17D vaccine safety and immunogenicity in the elderly. Hum Vaccin. 2005; 1(5):207-214; Black, et al. BCG-induced increase in interferon-gamma response to mycobacterial antigens and efficacy of BCG vaccination in Malawi and the UK: two randomised controlled studies. Lancet. 2002; 359(9315):1393-1401). Studies of YFV vaccines suggest differential sensitization due to exposure to environmental mycobacteria can alter vaccine responsiveness (Lalor, et al. BCG vaccination induces different cytokine profiles following infant BCG vaccination in the UK and Malawi. J Infect Dis. 2011; 204(7):1075-1085). Of note, several vaccine candidates use the YF-17D backbone as a vector for presentation of antigens from DENV, Japanese encephalitis virus, West Nile virus, and HIV (Bonaldo, et al. Recombinant yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 gag induces SIV-specific CD8+ T-cell responses in rhesus macaques. J Virol. 2010; 84(7):3699-3706; Guy, et al. Preclinical and clinical development of YFV 17D-based chimeric vaccines against dengue, West Nile and Japanese encephalitis viruses. Vaccine. 2010; 28(3):632-649; Martins, et al. Immunogenicity of seven new recombinant yellow fever viruses 17D expressing fragments of SIVmac239 Gag, Nef, and Vif in Indian rhesus macaques. PLoS One. 2013; 8(1):e54434).
However, the present art has been unable to provide a neutralizing vaccine for dengue virus. Accordingly, the present invention satisfies this unmet need, providing a chimeric protein directed at the E protein fusion loop to neutralize Dengue virus infections, without regard to serotype.